KR20230163924A - Method for producing a customized tumor organoid panel and an anti-cancer drug screening system using the panel prepared by the method - Google Patents
Method for producing a customized tumor organoid panel and an anti-cancer drug screening system using the panel prepared by the method Download PDFInfo
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- KR20230163924A KR20230163924A KR1020230029394A KR20230029394A KR20230163924A KR 20230163924 A KR20230163924 A KR 20230163924A KR 1020230029394 A KR1020230029394 A KR 1020230029394A KR 20230029394 A KR20230029394 A KR 20230029394A KR 20230163924 A KR20230163924 A KR 20230163924A
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Abstract
본 발명은 동결되어 있는 종양 오가노이드 컬럼을 유전적 정보를 기반으로 선택하여 다양한 종양 오가노이드 조합을 만들고, 이를 이용하여 약물을 평가하는 방법에 관한 것이다. 구체적으로 약물 평가에 적절한 크기로 96-웰 스트립 플레이트 (96-well strip plate)에 종양 오가노이드를 동결하였다가 해동하여 안정화한 후, 다양한 종양 오가노이드를 조합하여 대량으로 빠르게 약물 스크리닝을 할 수 있도록 하는 커스터마이징된 종양 오가노이드 패널의 제조 방법 및 상기 방법으로 제조된 패널을 이용한 항암제 스크리닝 시스템에 관한 것이다.
본 발명에 따른 커스터마이징된 종양 오가노이드 패널을 이용하여 약물 평가를 진행하는 경우 종양 오가노이드 성장 속도 차이에 의한 동시 평가의 어려움을 해결할 수 있고, 다양한 종양 오가노이드를 이용하여 평가할 수 있기 때문에 대량 스크리닝이 가능하다. 또한, 특정 직경의 종양 오가노이드를 동결 처리함으로써 종양 오가노이드의 생존율을 높힐 뿐만 아니라 보다 신속한 약물 평가가 가능하므로 항암제 스크리닝 방법으로써 유용하게 이용될 수 있다.The present invention relates to a method of selecting frozen tumor organoid columns based on genetic information to create various tumor organoid combinations and using them to evaluate drugs. Specifically, tumor organoids are frozen in a 96-well strip plate to a size appropriate for drug evaluation, then thawed and stabilized, and then various tumor organoids are combined to enable rapid drug screening in large quantities. It relates to a method for producing a customized tumor organoid panel and an anticancer drug screening system using the panel produced by the method.
When drug evaluation is performed using the customized tumor organoid panel according to the present invention, difficulties in simultaneous evaluation due to differences in tumor organoid growth rates can be solved, and mass screening can be performed because evaluation can be performed using various tumor organoids. possible. In addition, freezing treatment of tumor organoids of a specific diameter not only increases the survival rate of tumor organoids but also enables more rapid drug evaluation, making it useful as an anticancer drug screening method.
Description
본 발명은 동결되어 있는 종양 오가노이드 컬럼을 유전적 정보를 기반으로 선택하여 다양한 종양 오가노이드 조합을 만들고, 이를 이용하여 약물을 평가하는 방법에 관한 것이다. 구체적으로 약물 평가에 적절한 크기로 96-웰 스트립 플레이트 (96-well strip plate)에 종양 오가노이드를 동결하였다가 해동하여 안정화한 후, 다양한 종양 오가노이드를 조합하여 대량으로 빠르게 약물 스크리닝을 할 수 있도록 하는 커스터마이징된 종양 오가노이드 패널의 제조 방법 및 상기 방법으로 제조된 패널을 이용한 항암제 스크리닝 시스템에 관한 것이다.The present invention relates to a method of selecting frozen tumor organoid columns based on genetic information to create various tumor organoid combinations and using them to evaluate drugs. Specifically, tumor organoids are frozen in a 96-well strip plate to a size appropriate for drug evaluation, then thawed and stabilized, and then various tumor organoids are combined to enable rapid drug screening in large quantities. It relates to a method for producing a customized tumor organoid panel and an anticancer drug screening system using the panel produced by the method.
암은 세계적으로 높은 사망률을 보이며, 서구 사회에서는 심혈관 질환 다음으로 가장 일반적인 사망 원인이다. 특히, 식생활이 서구화되어 고지방식의 섭취가 일반화되고, 환경 오염 물질의 급격한 증가, 음주량의 증가 등으로 대장암, 유방암, 간암, 전립선암 등이 지속적으로 증가하는 추세에 있으며, 인구의 고령화와 더불어 흡연 인구의 증가 및 대기 오염으로 인해 폐암이 증가하고 있는 실정이다. 이러한 실정에서 암의 조기 예방 및 치료를 가능하게 하여 인간 건강의 증진, 건강한 삶의 질 향상 및 인류 보건 증진에 기여할 수 있는 항암 물질의 창출이 절실히 요구되고 있다.Cancer has a high mortality rate worldwide and is the most common cause of death in Western societies after cardiovascular disease. In particular, as dietary habits have become Westernized, high-fat diets have become common, and colon cancer, breast cancer, liver cancer, and prostate cancer are continuously increasing due to a rapid increase in environmental pollutants and increased drinking, and with the aging of the population. Lung cancer is on the rise due to the increase in the smoking population and air pollution. In this situation, there is an urgent need for the creation of anticancer substances that can contribute to the improvement of human health, healthy quality of life, and human health by enabling early prevention and treatment of cancer.
오가노이드 (Organoid)는 줄기세포로부터 자가 재생 및 자가 조직화를 통해 형성된 3차원 세포 집합체를 뜻한다. 생체 외에서 장기와 유사한 세포를 포함하여 구조를 이루며 해당 장기의 일부 기능이 재현될 수 있는 장기 유사체로 여겨진다. 기존의 2차원적인 세포 배양법보다 해당 조직의 복잡성 및 세포 간 상호작용을 보다 더 구현할 수 있고, 윤리적인 문제가 대두되는 동물실험보다 비용도 저렴하여 질병 모델링, 약물 독성/효능 평가 및 약물 탐색 연구에 활용되고 있다.Organoid refers to a three-dimensional cell aggregate formed through self-renewal and self-organization from stem cells. It is considered an organ analog that has a structure containing cells similar to an organ in vitro and can reproduce some of the functions of the organ. It can better embody the complexity of the tissue and interactions between cells than existing two-dimensional cell culture methods, and is less expensive than animal testing, which raises ethical issues, making it suitable for disease modeling, drug toxicity/efficacy evaluation, and drug discovery research. It is being utilized.
환자의 암 조직으로부터 형성된 종양 오가노이드(Tumor Organoid)는 각각의 성장 속도가 다르기 때문에, 동시에 다양한 종양 오가노이드를 이용하여 약물 평가를 진행하려고 할 경우, 사용하려는 모든 종양 오가노이드의 성장 속도를 맞춰 배양하기가 어렵다. 그래서 각각의 종양 오가노이드를 배양한 후, 성장 속도에 따라 약물 평가를 따로 진행해야 하는 어려움이 있다. Tumor organoids formed from a patient's cancer tissue each have a different growth rate, so when attempting to conduct drug evaluation using various tumor organoids at the same time, culture them to match the growth rate of all tumor organoids to be used. It's difficult to do. Therefore, there is a difficulty in cultivating each tumor organoid and then conducting drug evaluation separately depending on the growth rate.
현재, 한국공개특허 제2020-0055673호에서는 세포 배양용 캐리어를 이용하여 제조된 오가노이드 및 이를 이용한 약물 독성 평가방법을, 한국공개특허 제2022-0018946호에서는 췌장암 오가노이드의 제조 방법에 관하여 개시하고 있으나, 다양한 오가노이드를 동시에 평가하여 항암제를 선별하고 대량으로 스크리닝이 가능한 커스터마이징된 종양 오가노이드 패널을 제조 방법에 대해서는 개시된 바가 없다.Currently, Korean Patent Publication No. 2020-0055673 discloses organoids manufactured using a cell culture carrier and a drug toxicity evaluation method using the same, and Korean Patent Publication No. 2022-0018946 discloses a method for manufacturing pancreatic cancer organoids. However, there has been no disclosure regarding a method for manufacturing a customized tumor organoid panel that can simultaneously evaluate various organoids to select anticancer drugs and conduct mass screening.
본 발명의 목적은 (a) 암 환자로부터 유래된 종양 오가노이드를 배양하는 단계; (b) 상기 배양된 종양 오가노이드를 패널로 제조하는 단계; (c) 상기 제조된 종양 오가노이드 패널을 동결화하는 단계; 및 (d) 상기 동결화된 종양 오가노이드 패널을 해동한 후 조합하는 단계를 포함하는 커스터마이징된 종양 오가노이드 패널 제조 방법을 제공하기 위한 것이다.The object of the present invention is to (a) culture tumor organoids derived from cancer patients; (b) preparing the cultured tumor organoids into a panel; (c) freezing the prepared tumor organoid panel; and (d) thawing the frozen tumor organoid panel and then combining it to provide a method for manufacturing a customized tumor organoid panel.
또한, 본 발명의 다른 목적은 상기 방법에 의해 제조된 커스터마이징된 종양 오가노이드 패널을 제공하기 위한 것이다.In addition, another object of the present invention is to provide a customized tumor organoid panel prepared by the above method.
또한, 본 발명의 또 다른 목적은 상기 제조된 커스터마이징된 종양 오가노이드 패널을 이용한 항암제 스크리닝 방법을 제공하기 위한 것이다.In addition, another object of the present invention is to provide an anticancer drug screening method using the customized tumor organoid panel prepared above.
상기 목적을 달성하기 위하여, (a) 암 환자로부터 유래된 종양 오가노이드를 배양하는 단계; (b) 상기 배양된 종양 오가노이드를 패널로 제조하는 단계; (c) 상기 제조된 종양 오가노이드 패널을 동결화하는 단계; 및 (d) 상기 동결화된 종양 오가노이드 패널을 해동한 후 조합하는 단계를 포함하는 커스터마이징된 종양 오가노이드 패널 제조 방법을 제공한다.In order to achieve the above object, (a) culturing tumor organoids derived from cancer patients; (b) preparing the cultured tumor organoids into a panel; (c) freezing the prepared tumor organoid panel; and (d) thawing the frozen tumor organoid panel and then combining it.
또한, 본 발명의 다른 목적은 상기 방법에 의해 제조된 커스터마이징된 종양 오가노이드 패널을 제공한다.Additionally, another object of the present invention is to provide a customized tumor organoid panel prepared by the above method.
또한, 본 발명의 또 다른 목적은 상기 제조된 커스터마이징된 종양 오가노이드 패널을 이용한 항암제 스크리닝 방법을 제공한다.In addition, another object of the present invention is to provide a method for screening anticancer drugs using the customized tumor organoid panel prepared above.
본 발명에 따른 커스터마이징된 종양 오가노이드 패널을 이용하여 약물 평가를 진행하는 경우 종양 오가노이드 성장 속도 차이에 의한 동시 평가의 어려움을 해결할 수 있고, 다양한 종양 오가노이드를 이용하여 평가할 수 있기 때문에 대량 스크리닝이 가능하다. 또한, 특정 직경의 종양 오가노이드를 동결 처리함으로써 종양 오가노이드의 생존율을 높힐 뿐만 아니라 보다 신속한 약물 평가가 가능하므로 항암제 스크리닝 방법으로써 유용하게 이용될 수 있다.When drug evaluation is performed using the customized tumor organoid panel according to the present invention, difficulties in simultaneous evaluation due to differences in tumor organoid growth rates can be solved, and mass screening can be performed because evaluation can be performed using various tumor organoids. possible. In addition, freezing treatment of tumor organoids of a specific diameter not only increases the survival rate of tumor organoids but also enables more rapid drug evaluation, making it useful as an anticancer drug screening method.
도 1은 커스터마이징된 종양 오가노이드 패널의 제조 방법 및 이후 해동하여 항암제 스크리닝을 실시하는 과정을 나타낸 도이다.
도 2는 본 발명의 실시예에 따른 종양 오가노이드 패널을 동결하고 3개월이 지난 뒤 해동한 후, 오가노이드 직경에 따라 종양 오가노이드의 형태가 변화하는지 여부를 현미경으로 관찰한 결과를 나타낸 도이다.
도 3은 본 발명의 실시예에 따른 종양 오가노이드 패널을 동결하고 3개월이 지난 뒤 해동한 후, Calcein-AM과 EthD-1을 이용하여 종양 오가노이드 직경에 따른 생존율 차이를 측정한 결과를 나타낸 그래프이다.
도 4는 기존 동결 보존법과 커스터마이징된 종양 오가노이드 패널 동결법에서 약물 처리하기 전까지 배양한 기간을 비교한 결과를 그래프로 나타낸 도이다.
도 5는 폐암 종양 오가노이드에서 기존 동결 보존법과 커스터마이징된 종양 오가노이드 패널 동결법에 따라 항암 효능 평가를 실시한 결과를 그래프로 나타낸 도이다.
도 6은 대장암 종양 오가노이드에서 기존 동결 보존법과 커스터마이징된 종양 오가노이드 패널 동결법에 따라 항암 효능 평가를 실시한 결과를 그래프로 나타낸 도이다.
도 7은 간암의 종양 오가노이드에서 기존 동결 보존법과 커스터마이징된 종양 오가노이드 패널 동결법에 따라 항암 효능 평가를 실시한 결과를 그래프로 나타낸 도이다.
도 8은 폐암 종양 오가노이드에서 기존 동결 보존법과 커스터마이징된 종양 오가노이드 패널 동결법에 따라 항암 효능 평가를 실시한 결과를 토대로 상관관계를 분석한 결과를 나타낸 도이다.
도 9는 대장암 종양 오가노이드에서 기존 동결 보존법과 커스터마이징된 종양 오가노이드 패널 동결법에 따라 항암 효능 평가를 실시한 결과를 토대로 상관관계를 분석한 결과를 나타낸 도이다.
도 10은 간암의 종양 오가노이드에서 기존 동결 보존법과 커스터마이징된 종양 오가노이드 패널 동결법에 따라 항암 효능 평가를 실시한 결과를 토대로 상관관계를 분석한 결과를 나타낸 도이다.Figure 1 is a diagram showing the method of manufacturing a customized tumor organoid panel and the subsequent thawing process for anticancer drug screening.
Figure 2 is a diagram showing the results of microscopic observation of whether the shape of the tumor organoid changes depending on the organoid diameter after freezing the tumor organoid panel according to an embodiment of the present invention and thawing 3 months later. .
Figure 3 shows the results of measuring the difference in survival rate according to tumor organoid diameter using Calcein-AM and EthD-1 after freezing the tumor organoid panel according to an embodiment of the present invention and thawing 3 months later. It's a graph.
Figure 4 is a graph showing the results of comparing the culture period before drug treatment in the existing cryopreservation method and the customized tumor organoid panel freezing method.
Figure 5 is a graphical representation of the results of anticancer efficacy evaluation in lung cancer tumor organoids according to the existing cryopreservation method and the customized tumor organoid panel freezing method.
Figure 6 is a graphical representation of the results of anticancer efficacy evaluation in colon cancer tumor organoids according to the existing cryopreservation method and the customized tumor organoid panel freezing method.
Figure 7 is a graphical representation of the results of anticancer efficacy evaluation in liver cancer tumor organoids according to the existing cryopreservation method and the customized tumor organoid panel freezing method.
Figure 8 is a diagram showing the results of correlation analysis based on the results of anticancer efficacy evaluation in lung cancer tumor organoids according to the existing cryopreservation method and the customized tumor organoid panel freezing method.
Figure 9 is a diagram showing the results of correlation analysis based on the results of anticancer efficacy evaluation in colon cancer tumor organoids according to the existing cryopreservation method and the customized tumor organoid panel freezing method.
Figure 10 is a diagram showing the results of correlation analysis based on the results of anticancer efficacy evaluation in liver cancer tumor organoids according to the existing cryopreservation method and the customized tumor organoid panel freezing method.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
본 발명은, (a) 암 환자로부터 유래된 종양 오가노이드를 배양하는 단계; (b) 상기 배양된 종양 오가노이드를 패널로 제조하는 단계; (c) 상기 제조된 종양 오가노이드 패널을 동결화하는 단계; 및 (d) 상기 동결화된 종양 오가노이드 패널을 해동한 후 조합하는 단계를 포함하는 커스터마이징된 종양 오가노이드 패널 제조 방법을 제공한다.The present invention includes the steps of (a) culturing tumor organoids derived from a cancer patient; (b) preparing the cultured tumor organoids into a panel; (c) freezing the prepared tumor organoid panel; and (d) thawing the frozen tumor organoid panel and then combining it.
본 발명에 있어서, "오가노이드(organoid)"는 세포를 3차원적으로 배양하거나 재조합하여 제작한 3차원의 세포 구조체를 의미하며, "미니 장기" 또는 "유사 장기"라고도 불린다. 상기 오가노이드는 구체적으로 기관 또는 조직을 구성하는 여러 종류의 세포들 중 하나 이상의 세포 종류를 포함하며, 조직 또는 기관의 형태와 기능을 재현할 수 있어야 한다.In the present invention, “organoid” refers to a three-dimensional cell structure produced by three-dimensionally cultivating or recombining cells, and is also called a “mini organ” or “pseudo-organ.” The organoid specifically contains one or more cell types among several types of cells constituting an organ or tissue, and must be able to reproduce the form and function of the tissue or organ.
상기 오가노이드는 신약 개발, 질병 치료 및 인공장기 개발에 활용될 수 있다. 신약 개발 시 기존 약물 테스트는 동물실험에서 발견되지 않았던 부작용이 인간에게서는 발견되는 등 동물실험과 임상실험의 결과가 상이하다는 한계점이 있었다. 오가노이드를 통해 이러한 한계점을 극복할 수 있을 것으로 예상되며, 동물실험에 대한 윤리적 논란에서도 벗어날 수 있다. 또한, 환자의 세포를 배양하여 유사기관을 제작할 수 있기 때문에 개인 맞춤형 임상시험의 방법론으로 주목받고 있으며, 향후 개인 맞춤형 장기 개발 등에 활용될 수 있다.The organoids can be used to develop new drugs, treat diseases, and develop artificial organs. When developing new drugs, existing drug testing had the limitation that the results of animal testing and clinical testing were different, such as side effects that were not found in animal testing, but were found in humans. It is expected that these limitations can be overcome through organoids, and ethical controversies over animal testing can also be avoided. In addition, because similar organs can be produced by culturing the patient's cells, it is attracting attention as a methodology for personalized clinical trials, and can be used in the development of personalized organs in the future.
본 발명에 있어서, "세포 배양"은 생물체의 조직으로부터 분리된 세포를 배양하는 것을 의미하며, 세포의 종류에 따라 배지의 종류, 온도 조건, 배양액 등은 공지된 바의 방법을 따른다.In the present invention, “cell culture” refers to culturing cells isolated from tissues of an organism, and the type of medium, temperature conditions, culture medium, etc. according to the type of cell follow known methods.
상기 "암 환자로부터 유래된"의 의미는 암 환자로부터 수득된 생물학적 시료를 의미하는 것으로서, 예를 들면, 전혈(whole blood), 백혈구(leukocytes), 말초혈액 단핵 세포(peripheral blood mononuclear cells), 백혈구 연층(buffy coat), (혈장(plasma) 및 혈청(serum)을 포함하는) 혈액, 객담(sputum), 눈물(tears), 점액(mucus), 세비액(nasal washes), 비강 흡인물(nasal aspirate), 호흡(breath), 소변(urine), 정액(semen), 침(saliva), 복강 세척액(peritoneal washings), 골반내 유체액(pelvic fluids), 낭종액(cystic fluid), 뇌척수막 액(meningeal fluid), 양수(amniotic fluid), 선액(glandular fluid), 췌장액(pancreatic fluid), 림프액(lymph fluid), 흉수(pleural fluid), 유두 흡인물 (nipple aspirate), 기관지 흡인물(bronchial aspirate), 활액(synovial fluid), 관절 흡인물(jointaspirate), 기관 분비물(organ secretions), 세포(cell), 세포 추출물(cell extract) 또는 뇌척수액(cerebrospinal fluid)을 포함할 수 있으나, 이에 제한되는 것은 아니다. The meaning of “derived from a cancer patient” refers to a biological sample obtained from a cancer patient, for example, whole blood, leukocytes, peripheral blood mononuclear cells, white blood cells. Buffy coat, blood (including plasma and serum), sputum, tears, mucus, nasal washes, nasal aspirate ), breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid. ), amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid ( It may include, but is not limited to, synovial fluid, joint aspirate, organ secretions, cells, cell extract, or cerebrospinal fluid.
본 발명의 목적상 상기 생물학적 시료는 암 환자로부터 분리된 조직 또는 암 세포주일 수 있으나, 이에 제한되는 것은 아니다.For the purpose of the present invention, the biological sample may be tissue or cancer cell line isolated from a cancer patient, but is not limited thereto.
상기 암 세포는 모든 종류의 암 유래 세포를 의미하는 것으로, 예를 들어, 상기 암은 뇌종양, 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 뇌간종양, 두경부 종양, 후두암, 구인두암, 비강암, 부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 흉부종양, 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 음경암, 요도암, 전립선암, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암 또는 피부암을 포함하나, 이에 제한되는 것은 아니다.The cancer cells refer to all types of cancer-derived cells. For example, the cancer includes brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer. , nasal cavity cancer, paranasal cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, large intestine. Cancer includes, but is not limited to, anal cancer, bladder cancer, kidney cancer, penile cancer, urethral cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, or skin cancer.
상기 패널은 배양된 종양 오가노이드 다양하게 조합하여 구성할 수 있으며, 일 구체예에서는 96-웰 스트립 플레이트(96-well strip plate)에 8열 * 12 컬럼 (8 rows x 12 columns)으로 제조하였으나, 이에 제한되는 것은 아니며, 필요에 따라 그 열과 컬럼 수를 조절하여 제조할 수 있다.The panel can be composed of various combinations of cultured tumor organoids, and in one embodiment, it was prepared in 8 rows x 12 columns in a 96-well strip plate. It is not limited to this, and can be manufactured by adjusting the heat and number of columns as needed.
상기 제작된 각 컬럼은 플레이트로부터 손쉽게 분리할 수 있으며, 필요에 따라 다양한 형태로 조합하여 플레이트를 구성할 수 있다.Each of the manufactured columns can be easily separated from the plate, and the plate can be formed by combining them in various shapes as needed.
상기 패널은 액체질소 보관 조건에서도 손상이 발생하지 않는 모든 소재로 제작될 수 있으며, 예를 들어 폴리프로필렌(polypropylene) 등의 소재로 제작될 수 있으나, 이에 제한되지 않는다.The panel may be made of any material that does not cause damage even under liquid nitrogen storage conditions. For example, it may be made of a material such as polypropylene, but is not limited thereto.
상기 동결화 하는 단계는 일반적으로 수행되는 동결 방법으로 이루어질 수 있으며, 바람직하게는 완만동결법(slow freezing) 또는 유리화동결법(vitrification)으로 행해질 수 있으나, 이에 제한되는 것은 아니다.The freezing step may be performed by a commonly performed freezing method, preferably by slow freezing or vitrification, but is not limited thereto.
상기 종양 오가노이드는 직경이 200 μm 이하일 수 있으며, 바람직하게는 50 μm 내지 150 μm일 수 있으나, 이에 제한되는 것은 아니다.The tumor organoid may have a diameter of 200 μm or less, preferably 50 μm to 150 μm, but is not limited thereto.
본 발명의 다른 양태에서, 본 발명은 상기 커스터마이징된 종양 오가노이드 패널을 이용하여 항암 물질을 스크리닝하는 방법을 제공한다.In another aspect of the present invention, the present invention provides a method for screening anticancer substances using the customized tumor organoid panel.
상기 항암 물질을 스크리닝하는 방법은 커스터마이징된 종양 오가노이드 패널을 해동하는 단계; 상기 해동된 패널에 후보 물질을 처리하는 단계; 및 항암 활성을 측정하는 단계를 포함할 수 있다.The method for screening the anticancer substance includes thawing a customized tumor organoid panel; Processing candidate materials on the thawed panel; And it may include measuring anticancer activity.
본 발명에 있어서, 상기 "스크리닝"이란, 다양한 물질로 이루어진 후보군으로부터 목적하는 특정한 성질을 갖는 물질만을 특이적으로 선별해내는 것을 의미한다.In the present invention, “screening” means specifically selecting only substances with specific desired properties from a candidate group consisting of various substances.
본 발명에서, 상기 "후보물질"이란, 암 조직의 성장 또는 전이에 대해 억제 활성을 갖거나, 암 개시 세포의 사멸을 유도할 수 있는 것으로 예상되는 미지의 물질을 의미한다. 상기 후보물질은 화합물, 펩티드, 단백질, 항체 및 천연 추출물 등일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the “candidate substance” refers to an unknown substance expected to have inhibitory activity on the growth or metastasis of cancer tissue or to induce death of cancer initiating cells. The candidate materials may include compounds, peptides, proteins, antibodies, and natural extracts, but are not limited thereto.
본 발명에서, 상기 후보물질은 합성 또는 천연 화합물의 라이브러리, 생물학적 라이브러리, 공간 어드레서블 패러럴 고상 또는 액상 라이브러리(Spatially addressable parallel solid phase or solution phase libraries) 등을 통해 얻을 수 있으며, 이에 제한되는 것은 아니다.In the present invention, the candidate material can be obtained through libraries of synthetic or natural compounds, biological libraries, spatially addressable parallel solid phase or solution phase libraries, etc., but is not limited thereto. .
상기 해동된 패널에 시험 항암 물질을 처리하는 단계 및 항암 활성을 측정하는 단계는 시험 물질의 "항암 활성 또는 독성을 검사하는 방법"이며, 그 종류를 제한하지 않는다. The step of treating the thawed panel with a test anti-cancer substance and measuring the anti-cancer activity is a “method of testing the anti-cancer activity or toxicity” of the test substance, and the type is not limited.
실시예 1. 인간 유래 종양 오가노이드 제작Example 1. Production of human-derived tumor organoids
1-1. 인간 간암 오가노이드 성장 배지1-1. Human liver cancer organoid growth medium
인간 간암 오가노이드는 10 mM HEPES (Gibco), 1X GlutaMAXTM supplement (Gibco), 1X B-27TM supplement (Gibco), 1X N-2 supplement (Gibco), 1% 페니실린-스트렙토마이신 (Penicillin-Streptomycin)이 포함된 어드밴스드 DMEM/F-12 (Advanced DMEM/F-12)에 Gastrin 1 (Sigma), N-Acetyl-L-cystein (Sigma), Nicotinamide (Sigma), A83-01 (Sigma), 인간 섬유아 세포 재조합 성장 인자 (Recombinant human fibroblast growth factor; FGF)-10 (PeproTech), 인간 간 세포 재조합 성장 인자 (Recombinant human hepatocyte growth factor; HGF, PeproTech), 인간 표피 성장 인자 재조합 단백질 (Human epidermal growth factor recombinant protein; EGF, Gibco), 덱사메타손 (Dexamethason, Sigma)을 첨가하여 배양하였다.Human liver cancer organoids were supplemented with 10mM HEPES (Gibco), 1X GlutaMAX TM supplement (Gibco), 1X B-27 TM supplement (Gibco), 1X N-2 supplement (Gibco), 1% Penicillin-Streptomycin. Advanced DMEM/F-12 contains Gastrin 1 (Sigma), N-Acetyl-L-cystein (Sigma), Nicotinamide (Sigma), A83-01 (Sigma), and human fibroblasts. Recombinant human fibroblast growth factor (FGF)-10 (PeproTech), Recombinant human hepatocyte growth factor (HGF, PeproTech), Human epidermal growth factor recombinant protein ; EGF, Gibco) and dexamethasone (Dexamethason, Sigma) were added and cultured.
1-2. 인간 폐암 오가노이드 성장 배지1-2. Human Lung Cancer Organoid Growth Medium
인간 폐암 오가노이드는 1X B-27TM supplement (Gibco), 1X N-2 supplement (Gibco), 1% 페니실린-스트렙토마이신 (Penicillin-Streptomycin)이 포함된 DMEM/F-12에 인간 EGF 재조합 단백질 (Human EGF recombinant protein, Gibco), 인간 FGF 기본 (Human FGF basic, Gibco)을 첨가하여 배양하였다.Human lung cancer organoids were cultured in DMEM/F-12 containing 1X B-27 TM supplement (Gibco), 1X N-2 supplement (Gibco), and 1% Penicillin-Streptomycin (Human EGF recombinant protein). EGF recombinant protein (Gibco) and human FGF basic (Gibco) were added and cultured.
1-3. 인간 대장암 오가노이드 성장 배지1-3. Human Colon Cancer Organoid Growth Medium
인간 대장암 오가노이드는 1X B-27TM supplement (Gibco), Primocin (Invivogen), 1% 페니실린-스트렙토마이신 (Penicillin-Streptomycin)이 포함된 DMEM/F-12에 Gastrin 1 (Sigma), N-Acetyl-L-cystein (Sigma), Nicotinamide (Sigma), A83-01 (Sigma), 인간 EGF 재조합 단백질 (Human EGF recombinant protein, Gibco)를 첨가하여 배양하였다. Human colon cancer organoids were grown in DMEM/F-12 containing 1X B-27 TM supplement (Gibco), Primocin (Invivogen), 1% Penicillin-Streptomycin, Gastrin 1 (Sigma), and N-Acetyl. -L-cysteine (Sigma), Nicotinamide (Sigma), A83-01 (Sigma), and human EGF recombinant protein (Gibco) were added and cultured.
1-4. 인간 간암, 폐암 또는 대장암 오가노이드 제조1-4. Fabrication of human liver, lung, or colon cancer organoids
첨가물이 들어가지 않은 배지로 조직을 세척하고, 2X collagenase type II (Gibco)를 1:1 비율로 넣어 37℃ 회전 교반기 상에서 1시간 동안 인큐베이션하였다. 250 xg, 3분 동안 원심분리하고 상층액을 제거하여 첨가물이 들어가지 않은 배지로 세포를 현탁하였다. 100 μm strainer로 거른 후, 적혈구를 제거하고 원심분리하여 상층액을 제거하였다. The tissue was washed with a medium without additives, 2X collagenase type II (Gibco) was added at a 1:1 ratio, and incubated for 1 hour on a rotating shaker at 37°C. Centrifugation was performed at 250 x g for 3 minutes, the supernatant was removed, and the cells were suspended in medium without additives. After filtering with a 100 μm strainer, red blood cells were removed and the supernatant was removed by centrifugation.
세포를 계수하고 간암, 폐암 또는 대장암 성장 배지와 마트리겔 (Matrigel, Corning)을 1 : 2 비율로 섞어 세포와 잘 섞어준다. 플레이트(Plate) 중앙에 마트리겔에 섞은 세포를 떨어트리고 세포 배양기에 넣어 굳혀준 후, 배양 배지에 10 μM Y-27632 Dihydrochloride (Biogems)를 첨가하여, 세포 배양기 (5% CO2, 37℃)에서 배양하였다. 배양 시 4일마다 Y-27632 Dihydrochloride가 첨가되지 않은 배지로 교체해주며 배양하였다.Count the cells and mix liver cancer, lung cancer, or colon cancer growth medium and Matrigel (Corning) in a 1:2 ratio and mix well with the cells. Drop the cells mixed with Matrigel in the center of the plate and solidify them in a cell incubator. Then, 10 μM Y-27632 Dihydrochloride (Biogems) was added to the culture medium and cultured in a cell incubator (5% CO 2 , 37°C). Cultured. During culture, the medium was replaced with Y-27632 Dihydrochloride-free medium every 4 days.
실시예 2. 커스터마이징된 종양 오가노이드 패널 플레이트의 제작Example 2. Fabrication of customized tumor organoid panel plates
2-1. 종양 오가노이드 패널 제작2-1. Tumor organoid panel fabrication
상기 제작된 종양 오가노이드(간암, 폐암 또는 대장암) 뿐 아니라 다양한 종양 오가노이드를 패널에 구성하여 커스터마이징된 종양 오가노이드 패널 플레이트를 제작할 수 있다. In addition to the tumor organoids produced above (liver cancer, lung cancer, or colon cancer), a customized tumor organoid panel plate can be produced by constructing a panel of various tumor organoids.
상기 수득한 간암, 폐암, 또는 대장암 오가노이드를 96 웰 스트립 플레이트 (96-well strip plate, 8 rows x 12 columns)에서 항암 물질 스크리닝이 가능한 직경까지 배양하였다. 각 환자의 조직으로부터 제작한 오가노이드를 각 플레이트에 배양한 후, 동결하여 보관한다. 실험 목적에 따라 종양 오가노이드를 선택하고, 동결 중인 종양 오가노이드 플레이트에서 필요한 수만큼 컬럼을 꺼내 새로운 종양 오가노이드 패널을 구성하여 배양한다. 상기 배양한 종양 오가노이드를 안정화한 후, 항암 물질을 처리하여 스크리닝을 진행할 수 있다. 상기 패널 제조 과정을 나타낸 도면을 첨부하였다(도 1 참조).The obtained liver cancer, lung cancer, or colon cancer organoids were cultured in a 96-well strip plate (8 rows x 12 columns) to a diameter capable of screening for anticancer substances. Organoids produced from each patient's tissue are cultured on each plate, then frozen and stored. Tumor organoids are selected according to the purpose of the experiment, and the required number of columns are removed from the frozen tumor organoid plate to form a new tumor organoid panel and culture it. After stabilizing the cultured tumor organoids, screening can be performed by treating them with anticancer substances. A drawing showing the panel manufacturing process is attached (see Figure 1).
2-2. 종양 오가노이드의 동결2-2. Freezing of tumor organoids
상기 구성된 종양 오가노이드 패널 플레이트를 유리화동결법 (vitrification)을 이용하여 동결하였다. The tumor organoid panel plate constructed above was frozen using vitrification.
배양 중인 종양 오가노이드를 원심분리하여 수득한다. 인산완충식염수 (phosphate-buffered saline)를 이용하여 세척하고, 평형 용액 (equilibration solution)을 넣은 후, 상온에서 5분간 방치한다. 원심분리하여 평형 용액을 제거하고, 유리화동결 용액 (vitrification solution)을 넣은 후 액체질소 탱크에 바로 보관하였다. Tumor organoids in culture are obtained by centrifugation. Wash using phosphate-buffered saline, add equilibration solution, and leave at room temperature for 5 minutes. The equilibrium solution was removed by centrifugation, a vitrification solution was added, and the solution was immediately stored in a liquid nitrogen tank.
2-3. 동결과 해동에 따른 오가노이드 형태 및 생존율 분석2-3. Analysis of organoid morphology and survival rate according to freezing and thawing
오가노이드의 직경이 50 이상 ~ 100 이하 μm, 100 초과 ~ 150 이하 μm, 200 μm 이상 되도록 배양하여 각각을 동결한다. 동결 3개월, 12개월 후 해동하여 오가노이드의 형태가 유지되는지 현미경으로 관찰하고 생존율을 관찰하였으며, 그 결과를 도 2 및 도 3에 나타내었다. Cultivate the organoids to have a diameter of 50 or more to 100 μm, greater than 100 to 150 μm, or more than 200 μm, and freeze each. After 3 and 12 months of freezing, the organoids were thawed and observed under a microscope to see if the shape of the organoids was maintained, and the survival rate was observed. The results are shown in Figures 2 and 3.
도 2에 나타난 바와 같이, 직경이 200 μm 이상인 종양 오가노이드를 동결한 후 해동하였을 때, 다른 그룹에 비해 오가노이드의 형태가 온전하게 유지되지 않음을 확인할 수 있었다. 또한, 도 3에서와 같이 Calcein-AM과 EthD-1을 이용하여 오가노이드 생존율을 확인한 결과, 직경이 커질수록 죽은 세포 비율이 높아지며, 200 μm 이상의 오가노이드는 중심부까지 손상이 있음을 확인할 수 있었다 (data not shown). 상기 결과에 따라 오가노이드의 직경이 200 μm 미만으로 동결하였을 때, 해동 후 생존율을 증가시킬 수 있음을 확인할 수 있다.As shown in Figure 2, when tumor organoids with a diameter of 200 μm or more were frozen and then thawed, it was confirmed that the shape of the organoids was not maintained intact compared to other groups. In addition, as shown in Figure 3, as a result of checking the organoid survival rate using Calcein-AM and EthD-1, it was confirmed that the proportion of dead cells increased as the diameter increased, and that organoids larger than 200 μm had damage even to the center ( data not shown). According to the above results, it can be confirmed that when organoids are frozen with a diameter of less than 200 μm, the survival rate after thawing can be increased.
실시예 3. 항암제 스크리닝 방법Example 3. Anticancer drug screening method
3-1. 항암제 스크리닝 방법3-1. Anticancer drug screening method
상기 실시예 2에서 제조된 종양 오가노이드 패널 중, 환자 및 암종 별로 필요에 따라 커스터마이징하여 패널을 구성할 수 있다.Among the tumor organoid panels prepared in Example 2, the panel can be configured by customizing it as needed for each patient and cancer type.
도 1에 나타난 바와 같이, 여러 환자(A - L)로부터 수득하여 제조된 종양 오가노이드 중, 필요에 따라 종양 오가노이드를 선택하여 커스터마이징된 패널을 구성하였다. As shown in Figure 1, among the tumor organoids obtained and prepared from several patients (A - L), tumor organoids were selected as needed to construct a customized panel.
3-2. 종양 오가노이드 직경에 따른 스크리닝 차이 평가3-2. Evaluation of screening differences based on tumor organoid diameter
1) 배양기간의 차이1) Differences in incubation period
기존 동결 보존법과 커스터마이징된 종양 오가노이드 패널 동결법에서 약물 처리하기 전까지 배양한 기간을 비교한 결과를 도 4에 나타내었다. 그 결과 폐암, 대장암, 간암 세가지 종양 오가노이드에서 종양 커스터마이징된 오가노이드 패널 동결법이 기존 동결법보다 최소 평균 8.7일에서 최대 17.3일 정도 더 빠르게 약물처리 기간에 도달한 것을 확인할 수 있었다.The results of comparing the culture period before drug treatment in the existing cryopreservation method and the customized tumor organoid panel freezing method are shown in Figure 4. As a result, it was confirmed that the tumor-customized organoid panel freezing method reached the drug treatment period in three tumor organoids, lung cancer, colon cancer, and liver cancer, by a minimum of 8.7 days and a maximum of 17.3 days faster than the existing freezing method.
2) 항암 효능 평가 비교2) Comparison of anticancer efficacy evaluations
폐암, 대장암, 간암의 세가지 종양 오가노이드에서 기존 동결 보존법과 커스터마이징된 종양 오가노이드 패널 동결법에 따라 항암 효능 평가를 실시하여 상관관계를 분석한 결과를 도 5 내지 도 7에 나타내었다.The results of the correlation analysis by evaluating anticancer efficacy in three tumor organoids of lung cancer, colon cancer, and liver cancer according to the existing cryopreservation method and the customized tumor organoid panel freezing method are shown in Figures 5 to 7.
항암 효능 평가는 폐암의 경우 에토포시드(Etoposide), 도세탁셀(docetaxel), 비노렐빈(Vinorelbine)의 세가지 항암제로, 대장암의 경우 5-FU, 옥살리플라틴(Oxaliplatin), 이리노테칸(Irinotecan)의 세가지 항암제로, 간암의 경우 소라페닙(Sorafenib) 항암제로 실시하였다. 그 결과, 도 5 내지 도 7에서 나타나는 바와 같이, 기존 동결 보존법에 따른 종양 오가노이드 대비 본 발명의 동결법에 따라 제조된 종양 오가노이드 패널의 항암 효능이 유사한 효능 평가가 가능한 것으로 확인되었다.Anticancer efficacy evaluation was conducted using three anticancer drugs: Etoposide, docetaxel, and Vinorelbine for lung cancer, and three anticancer drugs: 5-FU, Oxaliplatin, and Irinotecan for colon cancer. , In the case of liver cancer, Sorafenib was used as an anticancer drug. As a result, as shown in Figures 5 to 7, it was confirmed that the anticancer efficacy of the tumor organoid panel prepared according to the freezing method of the present invention was similar to that of the tumor organoids prepared according to the existing cryopreservation method.
나아가, 이를 통계적으로 확인하기 위해 스피어만 순위 상관 테스트(Spearman's rank correlation test)를 실시하였다. 폐암에 관한 상관관계 분석 결과 (도 8), 대장암에 관한 상관관계 분석 결과 (도 9) 및 간암에 관한 상관관계 분석 결과 (도 10)에서 확인할 수 있는 바와 같이, r 값은 각각 폐암 0.835, 대장암 0.762, 간암 0.869이며, p값은 모두 <0.001으로 기존의 방법과 높은 상관관계가 있어 효능에 대한 문제가 없음을 통계적으로 확인하였다.Furthermore, Spearman's rank correlation test was performed to statistically confirm this. As can be seen from the correlation analysis results for lung cancer (FIG. 8), the correlation analysis results for colon cancer (FIG. 9), and the correlation analysis results for liver cancer (FIG. 10), the r values are 0.835 for lung cancer, respectively. Colon cancer was 0.762 and liver cancer was 0.869, and the p values were all <0.001, showing a high correlation with the existing method and statistically confirming that there was no problem with efficacy.
상기 실험 결과에서 확인할 수 있듯이, 본 발명에 따른 종양 오가노이드 패널은 배양기간이 현저하게 단축되며, 단기간에 대량으로 오가노이드를 배양할 수 있을 뿐 아니라 기존의 방법으로 제조된 오가노이드와 항암 효능 실험 등의 차이가 없어 항암 치료를 위한 항암제 스크리닝 방법에 유용하게 활용할 수 있음을 확인하였다.As can be seen from the above experimental results, the tumor organoid panel according to the present invention significantly shortens the culture period, and not only can large quantities of organoids be cultured in a short period of time, but also can be tested for anticancer efficacy with organoids manufactured by conventional methods. As there was no difference, it was confirmed that it can be usefully used in anticancer drug screening methods for anticancer treatment.
Claims (6)
(b) 상기 배양된 종양 오가노이드를 패널로 제조하는 단계;
(c) 상기 제조된 종양 오가노이드 패널을 동결화하는 단계; 및
(d) 상기 동결화된 종양 오가노이드 패널을 해동한 후 조합하는 단계를 포함하는 커스터마이징된 종양 오가노이드 패널 제조 방법.(a) culturing tumor organoids derived from a cancer patient;
(b) preparing the cultured tumor organoids into a panel;
(c) freezing the prepared tumor organoid panel; and
(d) A method for manufacturing a customized tumor organoid panel comprising the step of thawing and then combining the frozen tumor organoid panel.
상기 (a) 단계의 암은 뇌종양, 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종뇌간종양, 두경부 종양, 후두암, 구인두암, 비강암, 부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 흉부종양, 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 음경암, 요도암, 전립선암, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암 및 피부암으로 이루어진 군으로부터 선택된 것인, 커스터마이징된 종양 오가노이드 패널 제조 방법.According to paragraph 1,
Cancer in stage (a) includes brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, Thyroid cancer, thoracic tumor, lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, penile cancer, urethral cancer. , a method of manufacturing a customized tumor organoid panel selected from the group consisting of prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, and skin cancer.
상기 (c) 단계의 동결화하는 단계는, 완만동결법(slow freezing) 또는 유리화동결법(vitrification)을 이용하는 것을 특징으로 하는, 커스터마이징된 종양 오가노이드 패널 제조 방법.According to paragraph 1,
The method of manufacturing a customized tumor organoid panel, characterized in that the freezing step of step (c) uses slow freezing or vitrification.
상기 (c) 단계의 동결화하는 단계는, 종양 오가노이드의 직경이 50 μm 내지 150 μm인 것을 특징으로 하는, 커스터마이징된 종양 오가노이드 패널 제조 방법.According to paragraph 1,
In the freezing step of step (c), a customized tumor organoid panel production method is characterized in that the diameter of the tumor organoid is 50 μm to 150 μm.
(b) 상기 해동된 패널에 후보 물질을 처리하는 단계; 및
(c) 항암 활성을 측정하는 단계를 포함하는 항암제 스크리닝 방법.(a) thawing the customized tumor organoid panel according to item 5;
(b) treating the thawed panel with a candidate material; and
(c) An anticancer drug screening method comprising the step of measuring anticancer activity.
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